Mouse TNF alpha ELISA Kit

SKU:
MOFI00104
€299

Description

ELISA Kit Technical ManualMSDS

Mouse TNF-alpha (Tumor Necrosis Factor Alpha) ELISA Kit - Information

The Assay Genie Mouse TNF-alpha (Tumor Necrosis Factor Alpha) ELISA Kit can assay for Mouse TNF-alpha in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How do our Mouse TNF-alpha (Tumor Necrosis Factor Alpha) ELISA Kits Work?

The Assay Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, Assay Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.

At Assay Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound Mouse TNF-alpha is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.

Mouse TNF-alpha (Tumor Necrosis Factor Alpha) ELISA Kit Data

Product Code

MOFI00104

UniprotP06804
Alias

TNFalpha, Tumor Necrosis Factor Alpha, TNF-alpha, DIF, TNF-alpha, TNFA, TNFSF2

Detection method

Sandwich ELISA, Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of Mouse TNF-alpha concentrations in serum plasma and other biological fluids.

Size

96T

Range

31.25-2000pg/ml

Sensitivity

18.75pg/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of Mouse TNF-alpha and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse TNF-alpha in samples.

MatrixRecovery range(%)Average(%)
serum(n=5)85-10597
EDTA plasma(n=5)85-9991
UFH plasma(n=5)89-10296
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse TNF-alpha and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample1:21:41:8
serum(n=5)88-93%85-103%99-105%
EDTA plasma(n=5)82-98%89-98%96-98%
UFH plasma(n=5)80-94%83-100%83-100%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Mouse TNF-alpha (Tumor Necrosis Factor Alpha) ELISA Kit Protocol

The below protocol is a sample protocol for Mouse TNF-alpha (Tumor Necrosis Factor Alpha) ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISA Kits allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Mouse TNF-alpha Antibody present in their sample.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Assay Protocol:

1.Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3.Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
6.Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9.Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

Mouse TNF-alpha (Tumor Necrosis Factor Alpha) ELISA Kit components

96 Assays

Storage

ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5 -

Other materials and equipment required:

The Assay Genie Mouse TNF-alpha (Tumor Necrosis Factor Alpha) ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir

Sample Preparation

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample TypeProtocol

Serum

If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

Plasma

Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.

Urine & Cerebrospinal Fluid

Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.

Cell culture supernatant

Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.

Cell lysates

Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.

Tissue homogenates

The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.

Tissue lysates

Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.

Breast Milk

Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

Mouse TNF-alpha Protein Information

UniProt Protein Function:TNF-a: Cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. It is mainly secreted by macrophages and can induce cell death of certain tumor cell lines. It is potent pyrogen causing fever by direct action or by stimulation of interleukin-1 secretion and is implicated in the induction of cachexia, Under certain conditions it can stimulate cell proliferation and induce cell differentiation. Homotrimer. Interacts with SPPL2B. Belongs to the tumor necrosis factor family.
UniProt Protein Details:

Protein type:Motility/polarity/chemotaxis; Membrane protein, integral; Apoptosis; Cytokine

Cellular Component: extracellular space; cell surface; integral to plasma membrane; extracellular region; integral to membrane; lipid raft; secretory granule; recycling endosome; membrane; plasma membrane; intracellular; phagocytic cup; external side of plasma membrane

Molecular Function:identical protein binding; protein binding; protease binding; cytokine activity; tumor necrosis factor receptor binding

Biological Process: positive regulation of JNK activity; extracellular matrix organization and biogenesis; positive regulation of nitric oxide biosynthetic process; positive regulation of NFAT protein import into nucleus; activation of MAPK activity; positive regulation of osteoclast differentiation; positive regulation of apoptosis; positive regulation of transcription, DNA-dependent; multicellular organismal development; response to glucocorticoid stimulus; positive regulation of caspase activity; positive regulation of NF-kappaB import into nucleus; osteoclast differentiation; positive regulation of translational initiation by iron; positive regulation of membrane protein ectodomain proteolysis; activation of NF-kappaB transcription factor; positive regulation of MAP kinase activity; tumor necrosis factor-mediated signaling pathway; cellular extravasation; positive regulation of phagocytosis; negative regulation of interleukin-6 production; JNK cascade; negative regulation of osteoblast differentiation; positive regulation of action potential; regulation of immunoglobulin secretion; negative regulation of protein complex disassembly; positive regulation of cytokine production; positive regulation of heterotypic cell-cell adhesion; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of mitosis; response to virus; positive regulation of interleukin-6 production; positive regulation of interleukin-8 biosynthetic process; glucose metabolic process; positive regulation of chemokine production; negative regulation of cytokine secretion during immune response; positive regulation of protein transport; detection of mechanical stimulus involved in sensory perception of pain; cell activation; defense response to Gram-positive bacterium; organ morphogenesis; induction of apoptosis via death domain receptors; DNA damage response, signal transduction resulting in induction of apoptosis; defense response to bacterium; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; negative regulation of L-glutamate transport; negative regulation of transcription, DNA-dependent; leukocyte migration; sequestering of triacylglycerol; apoptosis; positive regulation of smooth muscle cell proliferation; positive regulation of JNK cascade; defense response; negative regulation of transcription from RNA polymerase II promoter; positive regulation of interleukin-18 production; signal transduction; chronic inflammatory response to antigenic stimulus; positive regulation of synaptic transmission; positive regulation of hair follicle development; negative regulation of cell proliferation; regulation of protein secretion; regulation of osteoclast differentiation; negative regulation of lipid catabolic process; positive regulation of neuron apoptosis; positive regulation of cell proliferation; lipopolysaccharide-mediated signaling pathway; protein kinase B signaling cascade; positive regulation of chronic inflammatory response to antigenic stimulus; regulation of I-kappaB kinase/NF-kappaB cascade; inflammatory response; caspase activation; positive regulation of humoral immune response mediated by circulating immunoglobulin; regulation of protein amino acid phosphorylation; positive regulation of protein complex disassembly; transformed cell apoptosis; calcium-mediated signaling; MAPKKK cascade; positive regulation of peptidyl-serine phosphorylation; humoral immune response; regulation of cell proliferation; positive regulation of protein kinase B signaling cascade; cell proliferation; positive regulation of interferon-gamma production; negative regulation of glucose import; positive regulation of programmed cell death; positive regulation of chemokine biosynthetic process; positive regulation of protein complex assembly; negative regulation of viral genome replication; protein import into nucleus, translocation; positive regulation of protein kinase activity; activation of MAPKKK activity; positive regulation of fever; immune response; positive regulation of protein amino acid phosphorylation; receptor biosynthetic process; negative regulation of myoblast differentiation; leukocyte tethering or rolling; regulation of insulin secretion; positive regulation of cytokine secretion; positive regulation of inflammatory response

NCBI Summary:This gene encodes a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor (TNF) superfamily. Members of this family are classified based on primary sequence, function, and structure. This protein is synthesized as a type-II transmembrane protein and is reported to be cleaved into products that exert distinct biological functions. It plays an important role in the innate immune response as well as regulating homeostasis but is also implicated in diseases of chronic inflammation. In mouse deficiency of this gene is associated with defects in response to bacterial infection, with defects in forming organized follicular dendritic cell networks and germinal centers, and with a lack of primary B cell follicles. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jun 2013]
UniProt Code:P06804
NCBI GenInfo Identifier:135935
NCBI Gene ID:21926
NCBI Accession:P06804.2
UniProt Secondary Accession:P06804,O35853, Q62326, Q91VF3,
UniProt Related Accession:P06804
Molecular Weight:25,896 Da
NCBI Full Name:Tumor necrosis factor
NCBI Synonym Full Names:tumor necrosis factor
NCBI Official Symbol:Tnf
NCBI Official Synonym Symbols:DIF; Tnfa; TNF-a; TNFSF2; Tnfsf1a; TNFalpha; TNF-alpha
NCBI Protein Information:tumor necrosis factor; cachectin; tumor necrosis factor-alpha; tumor necrosis factor ligand superfamily member 2
UniProt Protein Name:Tumor necrosis factor
UniProt Synonym Protein Names:Cachectin; TNF-alpha; Tumor necrosis factor ligand superfamily member 2; TNF-a
Protein Family:Tumor necrosis factor
UniProt Gene Name:Tnf
UniProt Entry Name:TNFA_MOUSE
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Additional Information

Product type:
ELISA
Reactivity:
Mouse
ELISA Type:
Sandwich
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